The objective of this proposal is to use the genetics in Saccharomyces cerevisiae for identifying the key factors that mediate BRCA1 function. Mutations in BRCA1 account for a large portion of inherited breast and ovarian cancers. While BRCA1 has been implicated in multiple nuclear processes, its exact function remains to be elucidated. The proposed study promises to uncover the essential components involved in the BRCA1-dependent pathway, thus providing potential targets for correcting the defects caused by BRCA1 mutations. In addition, the genetic methods developed in this study are likely to offer innovative approaches of diagnostic testing and drug screening. Preliminary data suggest that, when introduced into yeast, BRCA1 affects nuclear functions in yeast in a similar manner as it does in human cells. Importantly, multiple cancer-predisposing mutations in BRCA1 abolish its function in both organisms, thus validating the use of the yeast genetics to elucidate BRCA1 function. It is hypothesized that BRCA1 interacts with a highly conserved and essential factor(s) in eukaryotes, and that the yeast genetic approaches will provide insightful information in the design of new drugs specifically targeted at BRCA1-dependent cancers. The specific aims of the proposal are: 1) To determine the critical residues in BRCA1 that are responsible for growth inhibition and stimulation of plasmid stability in yeast. A series of cancer-predisposing mutations, neutral polymorphisms, and site-directed mutations will be generated by site-directed mutagenesis, and their effect on growth inhibition and plasmid stability will be analyzed. These experiments will strengthen the relationship between cancer susceptibility and the BRCA1 effect in yeast cells. Mutants generated from this study will also be valuable in characterizing the interaction between BRCA1 and the putative target proteins. 2) To use the growth inhibition assay and plasmid stability assay for identifying the target proteins that mediate BRCA1 function. The yeast growth inhibition assay will be used to isolate multi-copy suppressors from mammalian and yeast cDNA libraries that can suppress the growth inhibition by BRCA1. The plasmid stability assay will be used to screen for second-site mutations that can affect BRCA1 function in regulating DNA replication. Biochemical, molecular and cell biological approaches will be used to establish a direct relationship between BRCA1 and the putative target proteins.